This work aimed to elucidate the anti-inflammatory effects of ethyl acetate fraction from Cnidium officinale Makino with a cellular system of LPS-stimulated RAW 264.7 and THP-1 cells. Some key pro-inflammatory cytokines and mediators including NO, iNOS, PGE2, COX-2, TNF-α, NF-kB p50 and NF-kB p65 were studied by sandwich ELISA and western blot analysis. Ethyl acetate fraction could significantly inhibit the production of NO, PGE2, TNF-α, iNOS and COX-2 in LPS-stimulated cell than that of single LPS-stimulated. And ethyl acetate fraction suppresses the activation of NF-kB p50 and NF-kB p65. All the results showed that ethyl acetate fraction had a good anti-inflammatory effect on LPS-stimulated RAW264.7 and THP-1 cells. Taken together, the anti-inflammatory actions of ethyl acetate fraction from Cnidium officinale Makino might be due to the down-regulation of NO, PGE2, TNF-α, iNOS and COX-2 via the suppression of NF-kB activation.